221

Biochimica et Biophysica Acta, 385 (1975) 221--231 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27614

APPARENT S U L F A T I O N OF GLYCOSAMINOGLYCANS BY ASCORBIC ACID 2- [ 3 s S ] SUL FATE: AN E X P L A N A T I O N

STANLEY S. SHAPIRO and JAMES P. POON

Department of Biochemical Nutrition, Hoffmann-La Roche Inc., Nutley, N.J. 07110 (U.S.A.) (Received September 23rd, 1974)

Summary The sulfation of glycosaminoglycans b y ascorbic acid 2-[ 3 s s] sulfate was studied in costal cartilage and chondrocytes in vitro. Negligable (if any) sulfation of glycosaminogiycans was detected with immediately isolated ascorbic acid 2-[~SS]sulfate. However, formation of [3sS]giycosaminoglycans was readily detected with ascorbic acid 2-[35S]sulfate which had been stored at --20°C for several days. The [ 3 s S] glycosaminoglycans did n o t result from the direct transfer of 3 s S from ascorbic acid 2-sulfate b u t rather from a decomposition product of ascorbic acid 2-[ 3 s S] sulfate. Evidence is presented to show that the sulfation pathway with the decomposition product involves exchange with inorganic sulfate, and strongly suggests that sulfation proceeds via 3'-phosphoadenosine 5'-phosphosulfate. The decomposition p r o d u c t appears similar to inorganic sulfate in several test systems. In view of these observations, it is suggested that previous conclusions implicating ascorbic acid 2-sulfate as a biological sulfate donor, based on the use of ascorbic acid 2-[ 3 s S] sulfate be re-evaluated.

Introduction Ascorbic acid 2-sulfate was initially synthesized by Ford and R u o f f [1], w h o postulated that this derivative of ascorbic acid might function as an in vivo sulfating agent. The subsequent isolation of ascorbic acid 2-sulfate from brine shrimp [2,3], fish [4], rat organs [5], and human urine [6], lent further support to this hypothesis. Ascorbic acid 2-sulfate was postulated to play a role in the sulfation of giycosaminoglycans, and thereby elucidate one of the unknown mechanisms b y which ascorbic acid is involved in the maintenance of connective tissues. Conflicting reports on the antiscorbutic effect of ascorbic acid 2-sulfate in guinea pigs [7--9] as well as its role in the sulfation of cholesterol to choles-

222 terol sulfate [10,11] have confused the concept as to whether ascorbic acid 2-sulfate is an inactive metabolite of ascorbic acid or is in fact an active biological intermediate. All previous investigations concerning the role of ascorbic acid 2-sulfate in biological sulfating systems have utilized ascorbic acid 2-[ ~ s S] sulfate [10,12-15]. During our investigations on the possibility of ascorbic acid 2-[ * sS]sulfate functioning as a sulfate donor in glycosaminoglycan biosynthesis we observed an 3 SS_labeled decomposition product. It is believed that our findings clarify some of the confusion concerning the role of ascorbic acid 2-sulfate in biological sulfations in general, and in sulfation of glycosaminoglycans in particular. Experimental procedure

Systems o f ascorbic acid 2-[3SS]sulfate. Dipotassium ascorbic acid 2-[ 3 SS]sulfat e dihydrate (spec. act. 1.99 Ci/g) was generously supplied by Dr C. Perry and barium ascorbic acid 2-[3SS]sulfate (spec. act. 1.55 Ci/g) was generously supplied by Dr R. Muccino of Hoffmann-La Roche Inc. [3 SS] Glycosaminoglyca n biosynthesis with costal cartilage strips. Costal cartilage was removed from 160 g male rats and trimmed of connecting tissue. The cartilage was minced and incubated in Krebs-Ringer-bicarbonate for 4 h at 37°C under a mixture of O2/CO~ (95 : 5, v/v). The usual incubation mixture contained 20 mg cartilage, 1.3 pmol of 3 s SO ~- or ascorbic acid 2-[ .3s s] sulfate (spec. act. 63 Ci/mol) in 0.40 ml of buffer. MgC12 was substituted for MgSO4 in the Krebs-Ringer buffer to avoid isotope dilution o f the added radiolabeled material. Determination o f 3 sS incorporated into costal cartilage glycosaminoglycans. The incubation was terminated after 4 h b y rapidly withdrawing the media, and washing the cartilage twice with 3 ml of KC1. The washed cartilage was transferred from the KC1 to a 1.0-ml solution of 0.1 M acetate buffer, pH 5.5, containing 0.75 mg papain, 5 mM EDTA and 5 mM cysteine. The papain digestion was maintained at 57°C. After 18 h, 50% trichloroacetic acid was added to give a final trichloroacetic acid concentration of 10%. After dialysis overnight a 25-pl aliquot of the trichloroacetic acid supernatant solution containing the glycosaminoglycans was chromatographed on Whatmann No. 3 paper in isobutyric/0.5 M N H 4 O H (5 : 3, v/v) overnight [16]. The origins containing the radioactive glycosaminoglycans were cut out, placed in 10 ml of Aquasol (New England Nuclear) and counted in a liquid scintillation spectrophotometer. Preparation o f chondrocytes. Costal cartilage from 4-day-old rats were trimmed and cleaned of connecting tissue. The cartilage was sliced with an extra fine dissecting scalpel under a dissecting microscope. 200--400 mg of finely sliced cartilage was suspended in 5.0 ml of Pucks saline G containing 20 mg of collagenase (Worthington Biologicals) and gently stirred at 37°C for 3--4 h. The digest was passed through a stainless steel wire mesh to remove undigested debris. The chondrocytes were collected b y centrifugation at 1000 X g for 10 min at 10°C. The c h o n d r o c y t e pellet was washed by suspending, and recentrifuging the cells in 5.0 ml of growth media (Eagles basal media witk

223 Earles salts, 10% fetal calf serum and 100 units/ml of penicillin and streptomycin). The media contained MgC12 in lieu of MgSO4. The washing procedure was repeated four times. Media (3.0 ml) containing 106 cells were placed in Falcon tissue culture flasks (25 cm 2 growth area). The flasks were incubated at 37°C under 95% air and 5% CO2. After 18 h the media containing the nonadhering cells were decanted and fresh media added. 3 s SO 2- or ascorbic acid 2-[ 3 SS]sulfate was added at this time with fresh media. The usual addition contained 20 pCi of 35SO ~- or ascorbic acid 2-[ 3 KS]sulfate per 0.06 pmol of reagent. The incubation was terminated after 48 h by the addition of 1.5 ml of 1.0 M acetate buffer, pH 5.8, containing 1.0 mg papain, 5 mM EDTA, and 5 mM cysteine. The papain digestion was carried out at 55°C for 18 h. The [ 3 s S] glycosaminoglycans were determined as described above. Results 35 S incorporation into costal cartilage. Costal cartilage strips were incubated with 3 s SO ~- and ascorbic acid 2-[ 3 s S] sulfate in vitro as described under Experimental procedure. Both 3 s SO ~- and the ascorbic acid 2-[ 3 SS]sulfat e preparations functioned as sulfate donors during glycosaminoglycan biosynthesis in vitro. The [ 3 s S] glycosaminoglycans and proteoglycans produced with 35SO ~- and ascorbic acid 2-[ 3 SS]sulfate were compared by chondroitinase digestion and electrophoresis in several systems. There were no qualitative differences in the 3 SS.labele d macromolecules produced with either sulfate source. [3SS]glycosaminoglycan formation as a function o f ascorbic acid 2-[ 3 s S] sulfate concentration. When increasing quantities (constant specific activities) of ascorbic acid 2-[ 3 s S] sulfate are added to the incubation mixture, increased formation of [ 3 sS] glycosaminoglycan is observed (Table I). However, when the same experiment is performed with constant ascorbic acid 2-[ 3 SS]sulfate and increasing a m o u n t of unlabeled ascorbic acid 2-sulfate, strikingly different results are obtained (Table II). These data clearly show t h a t transfer of 3 s S from ascorbic acid 2-[ 3 sS] sulfate to glycosaminoglycans is TABLE I [35S]GLYCOSAMINOGLYCANS FATE

PRODUCED

WITH INCREASING

ASCORBIC ACID 2-[35S]SUL-

100 m g of c o s t a l cortilage w a s i n c u b a t e d u n d e r t h e standard c o n d i t i o n s as d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . T h e s p e c i f i c a c t i v i t y o f the ascorbic acid 2 - [ 3 5 S ] sulfate w a s 1 5 0 . 106 c p r n / ~ m o l . The radioa c t i v i t y of t h e [35S] g l y c o s a r n i n o g l y c a n r e p r e s e n t s a 20-pl a l i q u o t a p p l i e d to the c h r o m a t o g r a m f r o m a 1.0-ml s o l u t i o n . Ascorbic acid 2-[35S]sulfate (ktmol)

[35S] Glycosaminoglycans (cpm)

0.033 0.133 0.330 0.660 1.30

2 224 8074 12474 32316 71 0 3 2

224 T A B L E II [35S]GLYCOSAMINOGLYCANS 2-SULFATE

P R O D U C E D WITH I N C R E A S I N G U N L A B E L E D ASCORBIC ACID

20 m g o f costal cartilage w a s i n c u b a t e d u n d e r t h e s t a n d a r d c o n d i t i o n s as d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . E a c h r e a c t i o n c o n t a i n e d 0 . 0 6 p m o l o f a s c o r b i c acid 2-[35S] sulfate w i t h a specific a c t i v i t y of 3 0 3 - 106 c p m / ~ m o l . T h e [ 3 5 S ] g l y c o s a m i n o g l y c a n p r o d u c e d is e x p r e s s e d as a p e r c e n t a g e o f t h e radioa c t i v i t y i n c o r p o r a t e d w i t h o u t a d d e d u n l a b e l e d ascorbic acid 2-sulfate ( 2 0 3 2 0 0 c p m ) . A s c o r b i c acid 2-sulfate (pmol)

-

[35S] G l y c o s aminoglycan (%) 100 94 99.7 95.1 101 94.0 87.2

-

0.1 0.4 1.0 4.0 8.0 16.0

independent of the quantity of unlabeled ascorbic acid 2-sulfate added and strongly suggest that the 3 s S is transferred from a compound in the preparation that is not ascorbic acid 2-[ 3 s S] sulfate. Time d e p e n d e n t f o r m a t i o n o f a 3 SS.labele d c o m p o u n d from ascorbic acid 2 - [ 3 SS] sulfate. After synthesis and isolation, the ascorbic acid 2-[ 3SS]sulfate Ba r35S'j ascorbic acid 2-sulfate

80--

60

9 doys 40

20

0

u

8°f 1 60

jt

°°"

225 K2 ['35S] oscorbic acid 2 - s u l f o t e

'°°f t

J

6O

× t~

2 days 40

20

0 I00

9 ~h

80

60

8 months

2

4o

1 O

0 Fig. 1. T i m e d e p e n d e n t f o r m a t i o n o f t h e d e c o m p o s i t i o n p r o d u c t . T h e i n c r e a s e d a p p e a r a n c e o f t h e d e c o m p o s i t i o n p r o d u c t w a s f o l l o w e d o v e r a p e r i o d o f t i m e b y p e r i o d i c t h i n - l a y e r c h r o m a t o g r a p h y in e t h y l a c e t a t e / a c e t i c a c i d / w a t e r (4 : 3 : 3, v / v ) a n d s u b s e q u e n t r a d i o c h r o m a t o g r a p h y s c a n n i n g . A s c o r b i c acid 2 - s u l f a t e w a s l o c a l i z e d b y a n u l t r a v i o l e t q u e n c h s p o t ( d a r k a r e a ) . T h e a r r o w r e p r e s e n t s t h e r a d i o a c t i v i t y of the c o m p o u n d d e s i g n a t e d as t h e d e c o m p o s i t i o n p r o d u c t . ( A ) T h e i n c r e a s e d a p p e a r a n c e of t h e d e c o m p o s i t i o n p r o d u c t in a f r o z e n s o l u t i o n (5 m g / m l ) o f b a r i u m a s c o r b i c acid 2-[ 3 S S ] s u l f a t e o v e r a p e r i o d o f 22 days. (B) T h e i n c r e a s e d a p p e a r a n c e of t h e d e c o m p o s i t i o n p r o d u c t in a f r o z e n s o l u t i o n (5 m g / m l o f p o t a s s i u m a s c o r b i c acid 2-[ 35 S ] s u l f a t e o v e r a p e r i o d o f 8 m o n t h s ) .

was chromatographed in acetic acid/ethyl acetate/water (3 : 4 : 3, v/v) on cellulose F plates (E. Merck) and appeared essentially ~> 99% radiochemically pure (Fig. 1A). However, with the passing of time a new radiolabeled spot was detected on the cellulose plate after acetic acid/ethyl acetate/water chromatography (Fig. 1A). The new spot would eventually account for approx. 20% of the radioactive material within 3 months. This new product appears with potassium or barium salts or if the ascorbic acid 2-[ 3 sS] sulfate is stored as crystals at --20°C or in solution at --20°C (Fig. 1B). The increase in this unidentified product was followed and correlated with the increased sulfate donor activity of ascorbic acid 2-[ 3 sS] sulfate when incubated with chondrocytes (Table III). These observations strongly indicate the

226 T A B L E III

35S

INCORPORATION

BY C H O N D R O C Y T E S W I T H S T O R E D A S C O R B I C A C I D 2 - [ 3 5 S ] S U L F A T E

A s c o r b i c acid 2 - [ 3 5 S ] s u l f a t e (99 • 107 c p m ) w a s a d d e d to 3.0 m l of c u l t u r e m e d i a as d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . T h e [ 3 5 S ] g l y c o s a m i n o g l y c a n s p r o d u c e d is e x p r e s s e d as a p e r c e n t a g e of the r a d i o a c t i v i t y i n c o r p o r a t e d w i t h an e q u i v a l e n t q u a n t i t y o f 35SO 2-. Day's post synthesis of a s c o r b i e acid 2-[ 3S S ] sulfate

Relative p r o d u c t i o n of [ 35S] glycosaminoglycans (%)

4 27 59

1.8 7.7 8.5

°°EA80 °l 0

8°f

\

B

6o

ZJo 80F C

Fig. 2. Isolation of decomposition product and pure ascorbic acid 2-[35S]sutfate. A n aged sample of ascorbic acid 2-sulfate was chromatographed in ethyl acetate/acetic acid/water (4 : 3 : 3, v/v). T h e areas corresponding to ascorbic acid 2-sulfate and the decomposition product were eluted from the plate and collected. Aliquots of the collected samples were rechromatographed. (A) T h e radiochromatographic scan of the thin-layer chromatography of the aged ascorbic acid 2-[ 3 S S] sulfate. (B) T h e radiochromatographic scan of the isolated ascorbic acid 2-[3SS]sulfate. (C) T h e radiochromatographic scan of the isolated decomposition product. T h e q u e n c h spot (dark area) corresponds to ascorbic acid 2-sulphate. T h e arrow represents the radioactivity of the c o m p o u n d designated as the decomposition product.

227 involvement of this unidentified 35 S-labeled decomposition product in the formation of [ 35 S] glycosaminoglycans. [ 3 s S] Glycosaminoglycan formation from isolated decomposition product. The decomposition product was isolated b y elution from cellulose plates after chromatography in acetic acid/ethyl acetate/water. The eluate was concentrated under a stream of N 2 at room temperature. An aliquot of the eluted ascorbic acid 2-[3SS]sulfate and decomposition product were rechromatographed to measure the extent of purification. Fig. 2A illustrates the radiochromatographic separation of ascorbic acid 2-[ 3 s S] sulfate from the decomposition product. The decomposition product could not be completely removed from the ascorbic acid 2-[ 3 SS]sulfat e fraction (Fig. 2 B ) b y thin-layer chromatography. Fig. 2C shows that the decomposition product has been greatly enriched after elution from the plate b u t still contained detectable ascorbic acid 2-[ 3 sS] sulfate. The isolated decomposition product was tested for its ability to donate [ 3 SS]sulfat e to glycosaminoglycans (Table IV). A sample of ascorbic acid 2-[3SS]sulfate (10 • 106 cpm) prior to chromatography on cellulose plates resulted in formation of [ 3 s S] glycosaminoglycan. After isolation of the ascorbic acid 2-[ 3 SS] sulfate from the cellulose plates (Fig. 2 B ) a n d subsequent incubations, only 20% of the [ 35 S] glycosaminoglycan was formed (Table IV}. It should be noted that it was n o t possible to remove all the decomposition product from the ascorbic acid 2-[ 3 SS]sulfate. The isolated decomposition product contained essentially all of the sulfate donor activity. An enriched fraction of the decomposition product (3 " 106 cpm) resulted in approximately the same extent of [3~S]glycosaminoglycan produced as 10 • 106 cpm of unfractionated ascorbic acid 2-[ 35S]sulfate (Table IV). Similar results have been obtained with rat chrondrocytes. Characterization o f the 3 s S-labeled decomposition product. The purified 3 s S-labeled decomposition product was assayed for inorganic sulfate by t w o standard methods; the benzidine m e t h o d of Antonopoulos [ 1 7 ] , and the turbidity m e t h o d described b y Lloyd [ 1 8 ] . Approx. 90% of the radioactivity in the samples were identified as inorganic sulfate. There was no detectable inorganic sulfate in the unlabeled ascorbic acid 2-sulfate. TABLE IV [35S]GLYCOSAMINOGLYCANS PRODUCED WITH PURE DECOMPOSITION PRODUCT AND ASCORBIC ACID 2-[35S]SULFATE 20 m g of costal cartilage was incubated under the standard conditions as described under Experimental procedure. Reactions 1 and 2 contained 0.0625 #tool of ascorbic acid 2-sulfate. The [3SS] glycosaminoglycans produced is expressed as a percentage of Reaction 1 (aged ascorbic acid 2-[35S] sulfate).

35S s o u r c e

1. 2. 3. 4.

Ascorbic acid 2-[35S]sulfate Chromatographed ascorbic acid 2-[35S] sulfate 35S-labeled decomposition product Reactions 2 + 3

35S A d d i t i o n (epm)

[35S] Glycosaminoglycans (%)

10" 106 10 • 1 0 6 3 ' 106 13 • 1 0 6

100 19.6 81.2 108.6

228 TABLE V [35S]GLYCOSAMINOGLYCAN PRODUCTION WITH 35S-LABELED DECOMPOSITION IN T H E P R E S E N C E O F U N L A B E L E D A S C O R B I C A C I D 2 - S U L F A T E

PRODUCT

2 0 m g o f c o s t a l c a r t i l a g e w a s i n c u b a t e d u n d e r s t a n d a r d c o n d i t i o n s as d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . E a c h r e a c t i o n c o n t a i n e d 6 ' 106 c p m of 35S-labeled d e c o m p o s i t i o n p r o d u c t . T h e [ 3 5 S ] g l y c o s a m i n o g l y c a n p r o d u c e d is e x p r e s s e d as a p e r c e n t a g e of [35S ] g l y c o s a m i n o g l y c a n i n c o r p o r a t e d w i t h o u t added ascorbic acid 2-sulfate (683 050 cpm). Ascorbic acid 2-sulfate (pmol)

-

-

0.02 0.10 0.40 1.0 4.0 8.0 16.0

[35S] G l y c o s arainoglycans (%) 100 110 104 108 125 96 86 102

[a SS]glycosaminoglycans formation from the decomposition product in the presence of sulfate. The transfer of 3 s S from the decomposition product to glycosaminoglycans is independent of unlabeled ascorbic acid 2-sulfate (Table V) b u t is dependent on the presence of unlabeled inorganic sulfate (Table VI). Unlabeled inorganic sulfate is capable of diluting [ 3 s S] glycosaminoglycan formation. Thus, the decomposition product is in fact inorganic sulfate, or the sulfate transfer reaction proceeds via a free dissociable SO~-. Characterization of the sulfation pathway. Since the nature of the sulfating agent was not apparent, an a t t e m p t was made to distinguish the 3 s S transfer from the decomposition product to glycosaminoglycan with the known 3'-phosphoadenosine 5'-phosphosulfate pathway. Various inhibitors of 3'-phosphoadenosine 5'-phosphosulfate synthesis were incubated with cartilage under the standard conditions (Table VII). All the inhibitors tested, with the exception of SO23- and adenosine 5'-phosphosulfate, inhibited both systems equally. T A B L E VI [35S]GLYCOSAMINOGLYCAN PRODUCTION ENCE OF INORGANIC SULFATE

WITH DECOMPOSITION

PRODUCT

IN T H E P R E S -

2 0 m g o f c o s t a l c a r t i l a g e w a s i n c u b a t e d u n d e r t h e s t a n d a r d c o n d i t i o n s as d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . E a c h r e a c t i o n c o n t a i n e d 3 . 0 - 1 0 6 c p m o f d e c o m p o s i t i o n p r o d u c t . T h e [35S] g l y c o s a m i n o g l y c a n p r o d u c e d is e x p r e s s e d as a p e r c e n t a g e o f t h e r a d i o a c t i v i t y i n c o r p o r a t e d in the a b s e n c e of a d d e d sulfate (141300 cpm). Sulfate (/~mol)

-

-

0.4 4.0

[35S] G l y c o s aminoglycan (%) 100 12.7 1.3

229 T A B L E VII I N H I B I T I O N O F [35S] G L Y C O S A M I N O G L Y C A N

PRODUCTION

The reactions c o n t a i n e d 20 m g of caxtilage under the stand~trd i n c u b a t i o n c o n d i t i o n s described u n d e r E x p e r i m e n t a l p r o c e d u r e . T h e [ 3 5 S ] g l y c o s a m i n o g l y e a n s p r o d u c e d is e x p r e s s e d as a percentage o f 35S i n c o r p o r a t e d in the absence o f added inhibitors. Sulfate i n c o r p o r a t i o n into g l y c o s a m i n o g l y c a n s

Additions

(M)

None

35SO 2-

A s c o r b i c acid 2-{ 35S] sulfate

100

100

2 " 1 0 -3 4 1 0 -3 8 1 0 -3

93 83 63

85 75 52

Galactosamine UDP-glucuronic acid NaF EDTA Cyanide

10-1 lO-I 10-1 10-1 lO-I

100 100 100 100 64

100 100 100 100 50

Dinitrophenol rn-Chlorocarboxyl-cyanide Phenyl hydrazone

10-3 10-3

18 2.6

29 3.4

10-3 1 0 -3 10-2

94 54 45

86 57 45

10-2 10-2 10-2 10-5 5" 10-s 5- 10-3

100 82 65 100 97 93 59

100 83 70 98 66 19 3

5 • 1 0 -3 10-2

52 32

10 5

Pyrophosphate

Arsenate 5 ADP 5'-AMP 3'-AMP Adenosine phosphosulfate

10 ~

so -

SO~-, a competitive inhibitor of the ATP sulfurylase reaction, and adenosine 5'-phosphosulfate, the immediate precursor of 3'-phosphoadenosine 5'phosphosulfate inhibited the ascorbic acid 2-[ 3 s S] sulfate system to a greater extent than the 3 s SO 2- system. These results are consistent with the notion that in the ascorbic acid 2-[ 35 S] sulfate preparation, there is a low level impurity and this impurity is responsible for the 35 S transfer, and that the impurity is either inorganic sulfate, or a c o m p o u n d that readily exchanges with free inorganic sulfate. Thus, for a fixed amount of inhibitor, there would be greater inhibition in the incubation with less 35 S donor (the ascorbic acid 2-[ 3 sS]_ sulfate incubation}. Sulfation of 1-octanol by ascorbic acid 2-sulfate in the presence o f Br2. A "model organic system" has been described for the sulfation of alcohols by ascorbic acid 2-sulfate in the presence of mild oxidizing agents [ 1 9 , 2 0 ] . Ascorbic acid 2-[ 3 SS]sulfate, repurified aseorbic acid 2-[ 3 SS]sulfat e and purified decomposition product were used as reagents in this system. The purified decomposition product did not act like ascorbic acid 2-sulfate to sulfate 1-oc-

230 80

A

5s]-x

60

40

1

20

0

- B [35S~ascorbi c ocid 2-sulfote 80

60

i

~.

40 '1

20

J \_

,o

~ C

/l

~

'

35so

Fig. 3. S u l f a t i o n o f 1 - o e t a n o l . 1 - O e t y l s u l f a t e w a s g e n e r a t e d b y t h e p r e s e n c e o f s u l f a t e d o n o r a n d B r 2 as described by Mumma [19]. The reaction was ehromatographed on eeUulose plates in chloroform/ methanol/water/pyridine (65 : 50 : 8 : 0.5, v/v). The arrow corresponds to oetyl sulfate. The area of the origin corresponds to the sulfate donors tested. (A) The radioehromatographie scan of the reaction with isolated decomposition product. (B) The radioehromatographie scan of the reaction with isolated aseorbie a e l d 2-[ 3 S S ] s u l f a t e . ( C ) T h e r a d i o e h r o m a t o g r a p h i e s c a n o f t h e r e a c t i o n w i t h i n o r g a n i c 3 S S O~-.

tanol. The decomposition product resembled inorganic sulfate in this reaction, producing only trace amounts of 1-octyl sulfate (Fig. 3). Discussion Numerous investigators have reported the sulfation of macromolecules by 3 s S in biological systems. Bond [12,13] reported the sulfation of macromolecules by ascorbic acid 2-[ 3 SS]sulfate in human fibroblast in vitro. Campeau and March [14] reported that ascorbic acid 2-[ 3 SS]sulfate transferred 3 s S to macromolecules in rat liver slices in vitro. Halver et al. [15] utilizing the technique of autoradiography reported that 3 s S from ascorbic acid 2-[ 3 s S ] sulfate was fixed into the matrix of fish cartilage. Verlangieri and Mumma [10] postulated that ascorbic acid 2-[ 3 .~S] sulfate acting as a sulfating agent, sulfated cholesterol, resulting in the excretion of cholesterol sulfate. In none of the

231

above studies with ascorbic acid 2-[ 3 ‘S] sulfate was a 3 ‘S-labeled decomposition product described. Also, in most cases, the solvent systems used to assay ascorbic acid 2-t 3 5S] sulfate purity would not resolve the decomposition product from the parent compound. We have reported here that the radiochemically pure ascorbic acid 2-[ 3 ‘S] sulfate (2 99%) undergoes a chemical transformation (Fig. 1) which is probably mediated by P-decay of the 3 5S. We could not detect any of this material in stored unlabeled ascorbic acid 2-sulfate. The nature of this material is presently under investigation. The decomposition product is a sulfate donor in our test systems, migrates similarly to inorganic sulfate in several solvent systems, and after isolation from a thin-layer chromatographic plate reacts as inorganic sulfate in the benzidine and barium turbidity test. These results should alert future investigators studying the biological role of ascorbic acid 2-sulfate employing ascorbic acid 2-[ 3 5 S] sulfate to be wary of the stability of the substrate. The results presented, therefore, necessitate a re-evaluation of reports for biological sulfation by ascorbic acid 2-[ 3 ‘S] sulfate. Acknowledgments We wish to thank Dr C. Perry and Dr R. Muccino for the generous supply of ascorbic acid 2-sulfate. We also wish to acknowledge the suggestion of Dr C. Perry for the thin-layer chromatography system used to monitor the purity of ascorbic acid 2-[ 3 “S] sulfate. We also thank Drs M. Brin, J.J. Kamm, L. Machlin, and J. Hamilton for reviewing the manuscript. References 1 Ford, E.A. and Ruoff, P.M. (1965) Chem. Commun. 24.630431 2 Mead, C.G. and Finamore, F.J. (1969) Biochemistry 8, 2652-2655 3 Bond, A.D., McClelland, B.W.. Einstein, J.R. and Finamore, F.J. (1972) Arch. Biochem. Biophys. 153,207-214 4 Johnson, C.L.. Hammer, D.C.. Hslver, J.E. and Baker, E.M. (1971) Fed. Proc. 30. 1822 5 Mumma, R.O. and Verlangieri. A.J. (1972) Biochim. Biophys. Acta 273. 249-253 6 Baker. E.M., Hammer, D.C., March. S.C.. Tolbert, B.M. and Canham, J.E. (1971) Science 173, 826-827 7 Mumma, R.O.. McKee. E.E., Verlangieri, A.J. and Barron, G.P. (1972) Nutr. Rep. Int. 6,133-137 8 Kueniz, W., Avenia, R. and Kamm, J.J. (1974) J. Nutr. 140,952-956 9 Campeau. J.E., March, S.C. and Tolbert. B.M. (1973) Fed. Proc. 32,931 10 Verlangieri, A.J. and Mumma. R.O. (1973) Atherosclerosis 17,3748 11 Horn& D., Weber, F. and Wiss. 0. (1974) 2. KIin. Chem. Klin. Biochem. 2, 62-5 12 Bond, A.D. (1972) Fed. Proc. 31. 706 13 Bond, A.D. (1973) Fed. Proc. 32,932 14 Campeau, J.D. and March, S.C. (1972) Fed. Proc. 31. 705 15 HaIver, J.E., Johnson, C.L.. Smith, R.R.. Tolbert, B.M. and Baker, E.M. (1972) Fed. Proc. 31, 705 16 Suzuki. S. and Strominger, J.L. (1960) J. Biol. Chem. 235.257-266 17 Antonopoulos. C.A. (1962) Acta Chem. &and. 16.1521-1522 18 Lloyd, A.G. (1966) in Methods in Enzymology (Colowick, S.P. and Kaplan, N.O.. eds), Vol. VIII, P. 671. Academic Press, New York 19 Mumma, R.O. (1968) Biochim. Biophys. Acta 165. 571-573 20 Chu, T.M. and SIamwhite. Jr, W.R. (1968) Steroids 12, 309-321